Recombinant Varecia variegata Cytochrome c oxidase subunit 2 (MT-CO2)

What is Cytochrome c oxidase subunit 2 (MT-CO2) and what is its role in cellular metabolism?

MT-CO2 is one of the core subunits of mitochondrial Cytochrome c oxidase (CCO), the terminal enzyme of the electron transport chain in cellular respiration. It contains a dual core CuA active site critical for electron transfer function . This subunit plays a significant role in physiological processes by accepting electrons from cytochrome c and transferring them to oxygen, which is the final electron acceptor in the respiratory chain. This process is coupled to proton pumping across the inner mitochondrial membrane, contributing to the proton gradient that drives ATP synthesis. In Varecia variegata, as in other eukaryotes, MT-CO2 is encoded by the mitochondrial genome and is essential for mitochondrial function and cellular energy production.

How does Varecia variegata MT-CO2 compare to MT-CO2 from other species?

Comparative analysis reveals interesting evolutionary relationships between MT-CO2 proteins from different species. The table below summarizes key comparative features:

Mitochondrial genomes of closely related Varecia variegata and Varecia rubra share 96.8% nucleotide identity , indicating high conservation of mitochondrial genes including MT-CO2. The core functional domains, particularly the CuA binding site, remain conserved across species due to their critical role in electron transfer, while other regions show more variation reflecting evolutionary distance.

What expression systems are commonly used for producing recombinant Varecia variegata MT-CO2?

Based on the available research data, E. coli is the most commonly used expression system for producing recombinant Varecia variegata MT-CO2 . The methodological approach typically involves:

• Gene cloning into appropriate expression vectors (e.g., pET series)

• Transformation into E. coli expression strains (often DE3 strains)

• Induction with IPTG

• Expression with N-terminal His-tag for purification purposes

While E. coli is favored for its simplicity, rapid growth, and high protein yield, alternative expression systems may be considered depending on research objectives:

The choice of expression system should be guided by the specific research questions being addressed and the functional aspects of MT-CO2 being studied.

What are the optimal storage and handling conditions for recombinant Varecia variegata MT-CO2?

For optimal preservation of recombinant Varecia variegata MT-CO2 integrity and activity, the following storage and handling protocols are recommended based on research data :

Storage temperature:

• Long-term storage: -20°C to -80°C

• Working aliquots: 4°C for up to one week

Buffer composition:

• Tris-based buffer (typically Tris/PBS-based)

• 6% Trehalose or 50% glycerol

• pH 8.0

Reconstitution protocol:

• Briefly centrifuge lyophilized protein vial prior to opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration before aliquoting for long-term storage

Critical handling notes:

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Small volumes may become entrapped in the seal of the product vial during shipment and storage; brief centrifugation is recommended prior to opening

• Working aliquots should be stored at 4°C for no more than one week

• For extended experiments, prepare multiple small-volume aliquots rather than repeatedly accessing a single stock

What experimental approaches can be used to study the enzyme kinetics of recombinant Varecia variegata MT-CO2?

To study the enzyme kinetics of recombinant Varecia variegata MT-CO2, researchers can employ several complementary methodological approaches:

Spectrophotometric assays: These represent the gold standard for MT-CO2 kinetic analysis. Monitoring the oxidation of reduced cytochrome c spectrophotometrically at 550 nm provides real-time data on enzyme activity . The basic protocol involves:

• Preparation of reduced cytochrome c substrate

• Reaction initiation by adding purified MT-CO2

• Monitoring absorbance changes at 550 nm

• Calculation of initial reaction velocities at varying substrate concentrations

• Determination of kinetic parameters (Km, Vmax) using appropriate models (e.g., Michaelis-Menten)

Oxygen consumption measurements: Since MT-CO2 contributes to the reduction of oxygen to water, oxygen consumption rates provide direct measurement of enzyme activity. This can be measured using:

• Clark-type oxygen electrodes

• Optical oxygen sensors

• Fluorescence-based oxygen detection systems

Stopped-flow techniques: For rapid kinetics of electron transfer, stopped-flow spectroscopy provides millisecond resolution of reaction dynamics. This approach is particularly valuable for elucidating the electron transfer mechanism.

Inhibitor studies: As demonstrated in previous research with MT-CO2 from other species, compounds like allyl isothiocyanate (AITC) can influence activity . Inhibition studies can provide insights into:

• Binding sites

• Mechanism of action

• Structure-function relationships

For comprehensive kinetic analysis, experimental design should include:

How can I assess the functional integrity of recombinant Varecia variegata MT-CO2 after purification?

A multi-parameter approach is essential for comprehensive assessment of recombinant MT-CO2 functional integrity:

Activity assays:
The primary functional assessment involves measuring the protein's ability to catalyze cytochrome c oxidation. A standard activity assay protocol includes:

• Incubate purified recombinant MT-CO2 with reduced cytochrome c

• Monitor absorbance decrease at 550 nm

• Calculate activity in units of μmol cytochrome c oxidized per minute per mg protein

• Compare specific activity to reference standards or native enzyme preparations if available

Structural integrity assessment:

• SDS-PAGE and Western blotting: Confirm protein size and immunoreactivity. Expected molecular weight for His-tagged Varecia variegata MT-CO2 is approximately 26-27 kDa .

• Circular dichroism (CD) spectroscopy: Verify secondary structure content. MT-CO2 typically shows characteristic alpha-helical signatures with minima at 208 and 222 nm.

• Thermal shift assays: Determine protein stability and proper folding using differential scanning fluorimetry. A well-folded MT-CO2 should show a cooperative unfolding transition.

Binding assays:
Confirm the protein's ability to interact with its natural partners:

• Surface plasmon resonance (SPR): Measure binding kinetics with cytochrome c or other complex components

• Isothermal titration calorimetry (ITC): Quantify binding thermodynamics

• Co-immunoprecipitation: Verify interactions with other cytochrome oxidase subunits

Spectroscopic analysis:
The CuA center in MT-CO2 has characteristic spectroscopic properties:

• UV-visible spectroscopy: Look for characteristic absorption bands around 480-530 nm and 760-800 nm

• Electron paramagnetic resonance (EPR): Verify the integrity of the CuA center

A comprehensive assessment workflow should include:

What are potential applications of recombinant Varecia variegata MT-CO2 in evolutionary biology research?

Recombinant Varecia variegata MT-CO2 offers several valuable applications in evolutionary biology research:

Phylogenetic analysis and molecular dating:
MT-CO2 sequences provide valuable data for constructing phylogenetic trees and estimating divergence times, particularly within primate lineages. The close relationship between Varecia variegata and Varecia rubra (96.8% mitochondrial genome identity) demonstrates how MT-CO2 can help resolve relationships between closely related species.

Methodology:

• Sequence comparison of MT-CO2 across primate species

• Phylogenetic tree construction using maximum likelihood or Bayesian methods

• Molecular clock analysis to estimate divergence times

• Correlation with known geological or evolutionary events

Functional evolution studies:
Comparing the biochemical properties of recombinant MT-CO2 from different species can reveal functional adaptations to different ecological niches:

Mitochondrial-nuclear coevolution:
MT-CO2 interacts with nuclear-encoded subunits, making it an excellent model for studying mitonuclear coevolution:

• Express V. variegata MT-CO2 with nuclear-encoded subunits from different species

• Measure assembly efficiency and enzyme activity

• Identify compensatory mutations that maintain function

• Investigate the molecular basis of compatibility or incompatibility

Conservation biology applications:
As Varecia variegata is critically endangered , MT-CO2 studies can contribute to conservation efforts:

• Use MT-CO2 as a genetic marker for population studies

• Assess genetic diversity in wild and captive populations

• Identify unique adaptations that might require conservation

• Develop non-invasive sampling methods targeting MT-CO2 for monitoring wild populations

Reconstruction of ancestral sequences:
Using MT-CO2 sequences from extant species, researchers can:

• Infer ancestral MT-CO2 sequences

• Express reconstructed proteins in the laboratory

• Compare functional properties of ancestral and modern proteins

• Test hypotheses about the evolution of mitochondrial function

These applications demonstrate how recombinant Varecia variegata MT-CO2 can serve as a valuable tool for addressing fundamental questions in evolutionary biology while also contributing to conservation efforts for this critically endangered species.

What methods are most effective for studying protein-protein interactions involving MT-CO2?

Investigating protein-protein interactions involving MT-CO2 requires specialized approaches due to its membrane-associated nature. Based on research practices, the following methodologies are most effective:

Affinity-based methods:

• Co-immunoprecipitation (Co-IP): This approach leverages specific antibodies to pull down MT-CO2 along with its interaction partners.

  • Protocol outline:
    a. Solubilize membranes with mild detergents (digitonin or DDM)
    b. Incubate with anti-MT-CO2 antibodies or anti-tag antibodies for recombinant protein
    c. Capture complexes with protein A/G beads
    d. Wash and elute bound proteins
    e. Identify interacting proteins by mass spectrometry

• His-tag pull-down assays: Utilizing the His-tag on recombinant Varecia variegata MT-CO2 , researchers can:

  • Immobilize His-tagged MT-CO2 on Ni-NTA beads

  • Incubate with potential binding partners

  • Wash away non-specific binders

  • Elute and analyze bound proteins

Biophysical methods:

• Surface plasmon resonance (SPR): Provides real-time, label-free detection of protein-protein interactions with kinetic information.

  • Advantages: Determines kon and koff rates; no labeling required

  • Limitations: Requires immobilization which may affect membrane protein conformation

• Microscale thermophoresis (MST): Measures changes in movement of fluorescently labeled molecules in microscopic temperature gradients.

  • Advantages: Works in solution; requires small amounts of protein

  • Limitations: Requires fluorescent labeling

Structural methods:

In silico approaches:

Molecular docking has been successfully used with MT-CO2 from other species and can predict interaction sites before experimental validation.

Comparative effectiveness of methods for MT-CO2 interactions:

For optimal results, researchers should employ complementary approaches, beginning with computational predictions and affinity-based methods for interaction discovery, followed by biophysical methods for quantification and structural techniques for detailed characterization.

How can structural differences between Varecia variegata MT-CO2 and human MT-CO2 be leveraged for comparative studies?

Structural differences between Varecia variegata MT-CO2 and human MT-CO2 offer valuable opportunities for comparative studies that can reveal evolutionary adaptations and fundamental structure-function relationships. A systematic approach includes:

1. Sequence and structure comparison:

Begin by aligning the amino acid sequences of Varecia variegata MT-CO2 (227 amino acids) with human MT-CO2. Key elements to analyze include:

• Conservation in functional domains, particularly the CuA binding region

• Differential residues in transmembrane regions

• Species-specific insertions or deletions

• Patterns of conservation across different primate lineages

2. Homology modeling and structural analysis:

Using available crystal structures of cytochrome c oxidase as templates:

• Generate comparative homology models for both proteins

• Analyze differences in:

  • Secondary structure elements

  • Surface charge distribution

  • Ligand binding pockets

  • Interfaces with other subunits

3. Experimental verification through chimeric proteins:

Design and express chimeric proteins by swapping domains between lemur and human MT-CO2:

4. Functional comparative assays:

Test both proteins under identical conditions to identify functional differences:

• Enzyme kinetics across temperature ranges (20-40°C)

• pH dependence profiles (pH 6.0-8.5)

• Stability under oxidative stress conditions

• Interactions with species-specific variants of other subunits

5. Evolutionary adaptation analysis:

Correlate structural differences with ecological and physiological differences between species:

• Temperature adaptation (lemurs evolved in Madagascar's variable climate)

• Metabolic rate differences (lemurs have lower basal metabolic rates than humans)

• Diet adaptations (lemurs are primarily frugivorous)

6. Applications in protein engineering:

The comparative knowledge gained can be applied to:

• Design MT-CO2 variants with enhanced stability or activity

• Create temperature-adapted variants for biotechnological applications

• Develop species-specific inhibitors for research purposes

7. Methodological approach for a comprehensive comparative study:

• Express recombinant versions of both proteins using identical systems

• Perform parallel purification using standardized protocols

• Conduct structural analysis (CD spectroscopy, HDX-MS, limited proteolysis)

• Compare functional parameters under standardized conditions

• Test chimeric constructs to map structure-function relationships

• Correlate findings with evolutionary and ecological context

This systematic comparative approach not only advances our understanding of MT-CO2 evolution but also provides insights into mitochondrial adaptation across primates with potential applications in both basic research and conservation biology.

What are the challenges in expressing and purifying functional Varecia variegata MT-CO2, and how can they be addressed?

Expressing and purifying functional Varecia variegata MT-CO2 presents several technical challenges due to its nature as a mitochondrial membrane protein. Based on research literature and protein characteristics, here are the key challenges and evidence-based solutions:

Challenge 1: Membrane protein expression
MT-CO2 is a transmembrane protein, which typically shows poor expression and solubility in standard systems.

Solutions:

• Optimized E. coli strains: Use specialized strains like C41(DE3) or C43(DE3) designed for membrane protein expression

• Expression vector selection: Vectors with moderate promoter strength (like pET-28a) have been successfully used for MT-CO2 expression

• Expression conditions: Lower temperature (16-20°C) and reduced inducer concentration (0.1-0.5 mM IPTG) improve folding

• Fusion partners: Addition of solubility-enhancing tags like SUMO or MBP in addition to His-tag

Challenge 2: Protein solubilization and extraction
Extracting membrane proteins while maintaining native structure requires careful optimization.

Solutions:

• Detergent selection: Based on successful purification protocols for MT-CO2:

  Detergent	Concentration	Best For
  n-Dodecyl β-D-maltoside (DDM)	1-2%	Initial solubilization
  Digitonin	0.5-1%	Maintaining complex integrity
  Lauryl maltose neopentyl glycol (LMNG)	0.01-0.05%	Long-term stability

• Solubilization conditions: Gentle extraction at 4°C for 1-2 hours with constant gentle rotation

• Buffer optimization: Include glycerol (10-20%) and salt (150-300 mM NaCl) to enhance stability

Challenge 3: Protein yield
Membrane proteins typically express at lower levels than soluble proteins.

Solutions:

Challenge 4: Maintaining functionality
Ensuring the purified protein retains its native activity is particularly challenging.

Solutions:

• Cofactor supplementation: Include copper ions (Cu²⁺) during purification to maintain the CuA center

• Reconstitution strategies: After purification, reconstitute into nanodiscs or liposomes to provide a lipid environment

• Activity preservation: Include stabilizing agents in purification buffers:

  • 6% Trehalose

  • 50% Glycerol for storage

  • 1-5 mM DTT to prevent oxidation of critical cysteines

Challenge 5: Protein stability
As noted in search results , recombinant MT-CO2 can be sensitive to freeze-thaw cycles.

Solutions:

• Storage protocol: Store in small aliquots (50-100 μL) at -80°C

• Reconstitution guidance: Briefly centrifuge lyophilized protein prior to opening

• Handling recommendations: Maintain samples on ice; minimize time at room temperature

• Buffer composition: Tris-based buffer at pH 8.0 with 6% Trehalose has shown good stability

Challenge 6: Quality assessment
Verifying the functional integrity of purified MT-CO2 requires specialized approaches.

Solutions:

• Activity assays: Spectrophotometric measurement of cytochrome c oxidation

• Structural assessment: CD spectroscopy to confirm secondary structure integrity

• Purity standards: Aim for >90% purity as assessed by SDS-PAGE

• Homogeneity analysis: Size exclusion chromatography to verify monodispersity

By systematically addressing these challenges with the evidence-based solutions outlined above, researchers can significantly improve the likelihood of obtaining functional recombinant Varecia variegata MT-CO2 suitable for structural and functional studies.

How can post-translational modifications of recombinant Varecia variegata MT-CO2 be characterized?

Characterizing post-translational modifications (PTMs) of recombinant Varecia variegata MT-CO2 requires a multi-faceted analytical approach. Based on current research methodologies, the following comprehensive strategy is recommended:

1. Mass Spectrometry-Based Approaches:

Bottom-up proteomics: This is the primary approach for identifying and mapping PTMs.

• Protocol overview:

  • Digest purified MT-CO2 with proteases (typically trypsin, but also chymotrypsin for complementary coverage)

  • Separate peptides by nano-LC

  • Analyze by high-resolution MS/MS (e.g., Orbitrap or Q-TOF)

  • Search against databases with variable PTM options

  • Validate with manual spectrum interpretation

Top-down proteomics: Analyzing intact protein provides a comprehensive view of PTM combinations.

• Advantages: Preserves information about PTM co-occurrence patterns

• Challenges: More difficult for membrane proteins like MT-CO2

• Implementation: Direct infusion of purified protein into high-resolution mass spectrometer

Targeted PTM analysis: For known or suspected modifications:

2. Site-Specific Functional Assays:

For key PTMs identified by MS, site-directed mutagenesis can assess functional importance:

• Replace modified residues with non-modifiable variants

• Create phosphomimetic mutations (e.g., Ser→Asp for phosphorylation)

• Compare activity of wild-type and mutant proteins

3. Structural Characterization:

Hydrogen-deuterium exchange MS (HDX-MS): Provides information about how PTMs affect protein dynamics and solvent accessibility.

X-ray crystallography or Cryo-EM: If sufficient quantities of homogeneously modified protein can be obtained, these methods provide direct visualization of PTMs in the protein structure.

4. Expression System Considerations:

The choice of expression system significantly impacts the PTM profile:

5. Proteomic Comparison with Native Protein:

If available, comparing the PTM profile of recombinant protein with that of native MT-CO2 isolated from Varecia variegata tissue provides crucial validation:

• Extract mitochondria from tissue samples

• Isolate cytochrome c oxidase complex

• Analyze MT-CO2 subunit using identical proteomic workflows

• Compare modification sites and stoichiometry

6. PTM Workflow Integration:

A comprehensive characterization workflow should include:

• Initial broad PTM screening by LC-MS/MS

• Targeted analysis of identified modifications

• Functional assessment of key PTMs

• Structural characterization of PTM effects

• Comparison of PTM profiles across expression systems

7. Special Considerations for MT-CO2:

• CuA center coordination: The coordination of copper in the CuA center may involve PTMs of cysteine residues

• Transmembrane domain modifications: PTMs in transmembrane regions may require specialized extraction and analysis methods

• Low abundance modifications: Some functionally important PTMs may be present at low stoichiometry, requiring enrichment strategies

By implementing this comprehensive approach, researchers can obtain a detailed map of recombinant Varecia variegata MT-CO2 post-translational modifications, providing valuable insights into protein function and regulation.

What are the considerations for designing experiments to study the impact of mutations on MT-CO2 function?

Designing rigorous experiments to study the impact of mutations on Varecia variegata MT-CO2 function requires careful planning across multiple dimensions. Based on current research methodologies, here is a comprehensive framework:

1. Strategic Mutation Site Selection:

Begin with bioinformatic analysis to identify high-value targets:

• Sequence conservation analysis: Align MT-CO2 sequences across species ranging from closely related lemurs to distant mammals. Conservation scores can be calculated using methods like Jensen-Shannon divergence.

• Functional domain targeting: Based on MT-CO2's structure, prioritize:

  • CuA binding domain (critical for electron transfer)

  • Transmembrane regions (important for complex assembly)

  • Interaction interfaces with other COX subunits

  • Cytochrome c binding regions

• Disease-relevant sites: Include mutations analogous to human MT-CO2 variants associated with mitochondrial disorders

2. Mutation Design Strategy:

3. Expression and Purification Considerations:

• Use consistent expression and purification protocols for wild-type and mutant proteins to ensure comparability

• Express multiple mutants in parallel with wild-type controls

• Verify proper folding before functional assessment (using CD spectroscopy, thermal shift assays)

• Document protein yields as some mutations may affect expression efficiency

4. Comprehensive Functional Assessment:

Design a multi-parameter assessment protocol:

Primary activity assays:

• Spectrophotometric measurement of cytochrome c oxidation rates

• Oxygen consumption measurements

• Electron transfer kinetics using stopped-flow techniques

Structural stability measurements:

• Thermal denaturation profiles using differential scanning fluorimetry

• Chemical denaturation with urea or guanidinium chloride

• Limited proteolysis to assess conformational changes

Binding interaction studies:

• Surface plasmon resonance to measure cytochrome c binding kinetics

• Pull-down assays to assess interactions with other subunits

• Crosslinking combined with mass spectrometry to map interaction sites

5. Data Analysis Framework:

Establish rigorous data collection and analysis protocols:

• Collect dose-response curves across multiple substrate concentrations

• Determine enzyme kinetic parameters (Km, kcat, kcat/Km)

• Apply appropriate statistical tests (ANOVA with post-hoc tests)

• Use multiple replicates (minimum n=3) from independent protein preparations

6. Structure-Function Integration:

Correlate functional data with structural information:

• Map mutations onto 3D structural models

• Use molecular dynamics simulations to predict structural perturbations

• Calculate electrostatic surface potentials to understand charge distribution changes

7. Experimental Controls:

Include appropriate controls to strengthen experimental design:

8. Advanced Analytical Techniques:

For deeper mechanistic insights:

• EPR spectroscopy to assess changes in the CuA center

• Time-resolved spectroscopy to measure electron transfer rates

• Native mass spectrometry to analyze complex assembly

• Hydrogen-deuterium exchange to detect conformational changes

9. Experimental Design Example:

For a comprehensive study of the CuA binding domain in Varecia variegata MT-CO2:

• Identify conserved residues in the CuA binding domain

• Generate mutations: alanine substitutions, charge reversals, and conservative changes

• Express and purify all variants under identical conditions

• Assess copper binding by UV-visible and EPR spectroscopy

• Measure electron transfer kinetics using stopped-flow spectroscopy

• Determine complex assembly efficiency by BN-PAGE

• Test enzymatic activity across physiologically relevant temperatures (25-39°C)

• Correlate functional changes with structural predictions from molecular modeling

By implementing this comprehensive framework, researchers can generate robust insights into structure-function relationships in Varecia variegata MT-CO2, contributing to both fundamental understanding of cytochrome c oxidase function and evolutionary adaptations in lemur species.